Mutations in MeCP2, a methyI-CpG-binding protein that functions as a global transcriptional repressor, are a major cause of Rett Syndrome (RTT), an X-linked progressive neurological disorder. While the selective inactivation of MeCP2 in neurons is sufficient to confer a Rett-like phenotype in mice, the specific functions of MeCP2 in post-mitotic neurons are not known. We have found that MeCP2 binds to a site in BDNF promoter III just 3' to the site of transcriptional initiation and functions to repress expression of the BDNF gene. Membrane depolarization triggers the calcium-dependent phosphorylation and release of MeCP2 from the BDNF promoter, thereby facilitating BDNF promoter Ill-dependent transcription. These findings indicate that MeCP2 plays a key role in the control of activity-dependent gene expression and suggest that the deregulation of this process may underlie the pathology of RTT. To begin to test this hypothesis, we propose the following specific aims: 1) To characterize the sites of membrane depolarization/calcium-dependent MeCP2 phosphorylation. We will utilize a variety of methods to identify sites of activity-regulated phosphorylation on MeCP2 and develop phosphorylation site-specific antibodies to investigate the regulation of these modifications in cultured neurons and brain sections in response to a variety of stimulation protocols. 2) To assess the effect of phosphorylation on MeCP2 activity. Non-phosphorylatable mutant forms of MeCP2 will be generated and expressed in neuronal cultures to test the hypothesis that activity-induced MeCP2 phosphorylation is required for proper regulation of BDNF promoter activity as well as for neuronal processes such as synaptic development and maintenance. 3) To identify additional activity-regulated neuronal targets of MeCP2. Our findings raise the possibility that MeCP2 may be a general regulator of activity-dependent gene expression. We will employ a variety of techniques including gene expression profiling, RT-PCR, and chromatin immunoprecipitation to identify other activity-regulated targets of MeCP2 in post mitotic neurons. It is our hope that the proposed experiments will provide a better understanding of MeCP2 function, give insight into the mechanisms of activity-dependent gene expression, and provide new opportunities for the development of therapeutic strategies to alleviate RTT pathology.